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BIOLOGY  UBSART 


UPON   THE   FORMATION   OF   HYDROCHLORIC   ACID 

IN   THE   FOVEOL^E   AND    ON    THE   SURFACE    OF 

THE  GASTRIC  MUCOUS  MEMBRANE  AND  THE 

NON-ACID     CHARACTER     OF     THE    CON- 

TENTS  OF  GLAND  CELLS  AND  LUMINA. 


B.  C.  H.  HARVEY   AND   R.  R.  BENSLEY. 


[Reprinted  from  BIOLOGICAL  BULLETIN,  Vol.  XXIII.,  No.  4,  September,  1912.] 


(Reprinted  from  BIOLOGICAL  BULLETIN,  Vol.  XXIII.,  No.  4,  Sept.,  1912.] 


UPON   THE   FORMATION   OF   HYDROCHLORIC   ACID 
IN   THE    FOVEOL.E   AND    ON   THE   SURFACE   OF 
THE  GASTRIC  MUCOUS  MEMBRANE  AND  THE 
NON-ACID     CHARACTER     OF     THE    CON- 
TENTS OF  GLAND  CELLS  AND  LUMINA.1 

B.  C.  H.  HARVEY  AND   R.  RVB'EN&LIiY. 

CONTENTS, 

Introduction .'  1 ' .  225 

Facts  which  Have  Suggested  Some  Association  between  the  Parietal  Cells  and 

the  Hydrochloric  Acid  or  its  Antecedents 226 

The  Prussian  Blue  Reaction 227 

Fitzgerald's  Paper 228 

Our  Own  Experiments 230 

Summary  of  the  Results  Obtained  by  the  Prussian  Blue  Reaction 

and  their  Significance 237 

Experiments  with  Indicators:  The  Reaction  of  the  Secretion  within  the  Lumen 
of  the  Active  Fundus  Gland  and  within  the  Intracellular  Canaliculi  of 

the  Parietal  Cells 239 

Confirmatory  Tests 245 

The  Consistence  of  the  Secretion  in  the  Giand  Lumen 247 

Other  Considerations •. .  248 

Bibliography 248 

INTRODUCTION. 

Ever  since  it  was  discovered  that  hydrochloric  acid  was  pro- 
duced by  the  stomach,  investigators  have  been  interested  in  deter- 
mining the  mode  and  place  of  its  formation.  Is  this  acid  pro- 
duced as  such  by  the  gastric  glands  and  even  by  particular  cells 
of  these  glands,  or  do  the  glands  produce  only  chemical  sub- 
stances which  are  not  themselves  acid  but  which,  interacting  in 
the  foveolse  or  on  the  surface  of  the  mucous  membrane,  produce 
there  for  the  first  time  the  acid  as  such? 

Among  those  who  have  sought  to  discover  the  origin  of  this 
acid,  Claude  Bernard,  Briicke,  Lepine,  Trinkler,  Gmelin  and 
Oppel  were  unable  to  find  it  definitely  localized  in  the  glands. 

The  results  of  our  investigation  demonstrate  that  only  non- 
acid  substances  are  formed  by  the  glands,  and  that  the  contents 
of  the  gland  cells  and  lumina  are  not  acid,  even  when  hydro- 

1  From  the  Hull  Laboratory  of  Anatomy,  University  of  Chicago. 

225 


226  B.    C.   H.   HARVEY  AND   R.    R.    BENSLEY. 

chloric  acid  is  being  actively  formed  on  the  surface  of  the  mucous 
membrane. 

In  the  first  part  of  this  paper  we  shall  examine  very  briefly  the 
evidence  which  has  led  some  to  think  that  free  hydrochloric 
acid  is  formed  in  the  parietal  cells.  We  shall  consider  especially 
the  Prussian  blue  reaction,  which,  as  used  by  Miss  Fitzgerald 
(*io),  has  given  by  far  the  most  definite  results.  We  shall 
report |some  refits',  ^Ijich  we  have  obtained  by  this  method, 
wrhich  in  connection  with 'those  reported  by  her  show  that  this 
rea£ti$4/$0Q£jfo<ti  prov^'ihe  presence  of  free  hydrochloric  acid 
in  the  gastric  glands  under  normal  conditions. 

In  the  second  part  of  the  paper  we  shall  show  that  the  contents 
of  the  parietal  cells  and  of  the  lumina  of  the  gastric  glands  are 
not  acid  but  neutral  or  alkaline,  even  when  hydrochloric  acid 
is  being  actively  formed  on  the  surface  of  the  mucous  membrane. 

FACTS  WHICH   HAVE  SUGGESTED   SOME  ASSOCIATION   BETWEEN 

THE  PARIETAL  CELLS  AND  THE  HYDROCHLORIC  ACID 

OR   ITS  ANTECEDENTS. 

Heidenhain  ('70),  Langley  ('81)  and  others  were  able  to  asso- 
ciate other  secretory  functions  of  the  gastric  mucous  membrane 
very  definitely  with  other  cells.  This  left  the  parietal  cells  and 
the  formation  of  hydrochloric  acid.  Further,  Miss  Greenwood 
('85),  and  afterward  Macallum  ('08)  and  Miss  Fitzgerald  ('10), 
have  shown  that  chlorides  are  more  abundant  in  the  parietal 
cells  than  in  other  parts  of  the  gland.  These  facts  suggest  that 
there  is  some  association  between  the  formation  of  the  hydro- 
chloric acid  and  the  parietal  cells.  They  leave  the  question 
quite  open,  however,  as  to  where  the  free  hydrochloric  acid  is 
first  formed  as  such.  The  parietal  cells  probably  form  substances 
which  later  furnish  the  chlorine  of  the  hydrochloric  acid,  but  our 
results  appearing  in  the  second  part  of  this  paper  show  that  these 
cells  do  not  normally  contain  the  hydrochloric  acid  itself. 

Miss  Fitzgerald  ('10)  employed  the  Prussian  blue  reaction  and 
obtained  very  definite  results  which  must  be  carefully  considered 
in  the  decision  of  this  question.  She  found  the  Prussian  blue 
deposited  in  the  canaliculi  of  some  parietal  cells.  It  will  be 
worth  while  to  examine  this  reaction  and  the  varying  results 
which  have  been  obtained  with  it. 


FORMATION  OF  HYDROCHLORIC  ACID  IN  THE  FOVEOL/E.  227 

THE  PRUSSIAN  BLUE  REACTION. 

This  was  first  employed  by  Claude  Bernard  ('59)  in  his  classic 
experiment.  A  translation  of  his  account  is  as  follows:  "In  a 
rabbit  which  had  eaten  very  little  there  was  injected  into  the 
jugular  vein  a  solution  of  lactate  of  iron  and  then  a  solution  of 
prussiate  of  potassium;  both  solutions  were  warm.  Three  quar- 
ters of  an  hour  afterward  the  animal  was  killed  and  at  the  autopsy 
it  was  impossible  to  demonstrate  the  blue  color  in  the  tissue  of 
any  organ.  The  urine,  which  was  alkaline  and  cloudy,  was  not 
blue,  although  it  contained  both  prussiate  of  potassium  and  the 
iron  lactate,  for  it  sufficed  to  add  a  few  drops  of  hydrochloric  or 
sulphuric  acid  to  cause  the  blue  color  of  Prussian  blue  to  appear 
immediately.  Upon  opening  the  alimentary  canal  a  blue  color 
was  found  on  the  surface  of  the  mucous  membrane  of  the 
stomach  and  particularly  on  the  part  which  corresponded  to 
the  lesser  curvature  of  that  organ.  But  this  blue  was  quite 
superficial;  the  little  deposits  of  Prussian  blue  were  only  on  the 
surface  of  the  mucous  membrane,  and  a  microscopic  examination 
did  not  reveal  any  Prussian  blue  in  the  gastric  glands." 

Later,  Claude  Bernard  ('77)  said:  "The  acid  of  the  gastric 
juice  is  formed  only  after  the  secretion  of  the  juice,  the  glands 
secreting  a  liquid  which  breaks  up  into  an  acid  fluid  and  another 
product  as  yet  not  definitely  determined." 

The  results  which  we  have  to  report  indicate  that  the  decision 
reached  by  the  great  French  physiologist  is  correct. 

The  Prussian  blue  reaction  was  employed  also  by  Lepine  ('72) 
in  dogs.  He  used  potassium  ferrocyanide  with  lactate  of  iron  or 
sulphate  of  iron.  He  was  unable  to  obtain  Prussian  blue  in  any 
cells  of  the  gastric  gland  either  by  injection,  maceration,  or  by 
passing  the  salts  through  a  dialyzing  membrane  made  of  the 
gastric  mucous  membrane,  although  by  the  latter  method  he  did 
obtain  a  little  blue  in  a  lymphatic  space  of  the  connective  tissue 
between  the  glands.  He  concluded  that  the  acid  wras  not  formed 
as  such  within  the  gland. 

Sehrwald  ('89)  put  pieces  of  the  gastric  mucous  membrane 
into  a  solution  of  lactate  of  iron  for  one  day  and  later  into  a 
solution  of  potassium  ferricyanide.  He  believed  that  in  this 
way  he  would  obtain  a  deposit  of  Prussian  blue  at  the  seat  of 


228  B.    C.   H.   HARVEY   AND   R.    R.    BENSLEY. 

formation  of  the  acid.  His  results  were  not  definite,  although 
he  thought  the  parietal  cells  showed  more  blue  than  other 
parts  of  the  mucous  membrane.  The  experiment  was  repeated 
by  Miss  Fitzgerald,  who  found  the  results  too  indefinite  to 
decide  the  question,  and  by  ourselves  with  the  same  outcome. 

Fitzgerald's  Paper. 

Miss  Fitzgerald  ('10)  conducted  very  careful  and  elaborate 
experiments  by  this  method  and  has  obtained  by  far  the  most 
definite  results.  Her  experiments  and  the  results  are  set  forth 
in  the  following  table,  which  is  copied  from  her  paper:1 

We  would  like  to  direct  especial  attention  to  a  few  facts  reported 
in  this  table  and  in  her  paper.  First,  the  plates  accompanying 
her  paper  show  very  clearly  the  deposit  of  Prussian  blue  within 
the  canaliculi  and  in  other  parts  of  some  parietal  cells  and  also 
in  the  interglandular  blood  and  lymph  vessels,  and  in  wandering 
cells  and  leucocytes.  Second,  in  some  experiments  the  Prussian 
blue  reaction  was  not  obtained.  Third,  when  it  did  appear  it 
was  obtained  in  only  one  part  of  the  stomach,  namely,  that  near 
the  oesophagus  and  along  the  lesser  curvature.  This  is  not  the 
part  which  contains  parietal  cells  in  greatest  abundance.  Fourth, 
even  in  this  region  only  a  few  of  the  parietal  cells  showed  the 
Prussian  blue.  Those  of  the  deeper  third  of  the  gland  tubules, 
that  is,  the  third  farthest  from  the  free  surface  never  contained  it; 
and  in  that  part  of  the  gland  tubule  where  it  did  appear  it  was 
found  in  only  a  fraction  of  the  parietal  cells,  so  that  altogether 
she  found  it  in  only  a  small  percentage  of  the  total  number  of 
parietal  cells  of  the  stomach,  and  this  notwithstanding  the  fact 
that  during  some  part  of  the  long  time  the  experiments  lasted 
all  parts  of  the  mucous  membrane  must  have  been  in  full  digestive 
activity.  Fifth,  it  appeared  in  other  structures  as  well  as 
in  the  parietal  cells,  namely,  in  the  blood  vessels,  in  the  connective 
tissue  spaces  and  lymphatic  vessels,  in  wandering  cells,  and  in 
leucocytes.  Sixth,  in  two  instances  (rabbits  5  and  6)  it  did 
not  appear  in  the  canaliculi  of  the  parietal  cells  but  only  at  the 
surface  of  the  latter  remote  from  the  lumen  and  next  to  the  blood 
vessels.  » 

Our  own  results  have  confirmed  these  facts  entirely. 


FORMATION   OF   HYDROCHLORIC   ACID   IN   THE   FOVEOL/E.    229 


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23O  B.    C.    H.    HARVEY   AND    R.    R.    BENSLEY. 

From  these  results  Miss  Fitzgerald  concludes  that  the  hydro- 
chloric acid  was  formed  as  such  in  the  parietal  cells  and  secreted 
into  the  gland  lumina.  She  says  (p.  82) :  "The  occurrence  of  the 
Prussian  blue  reaction  in  the  canaliculi  of  the  parietal  cells  of  an 
animal  injected  with  a  solution  of  these  two  salts  affords  con- 
clusive evidence  of  the  presence  of  free  acid  within  these  struc- 
tures." 

The  absence  of  the  Prussian  blue  from  most  of  the  parietal  cells 
might  be  regarded  as  evidence  that  only  a  few  of  these  cells  are 
engaged  in  the  formation  of  hydrochloric  acid,  and  that  the 
greater  part  of  them  do  not  form  it.  She  explains  this  absence 
from  mo&t  of  the  parietal  cells  by  saying  that  it  may  have  been 
washed  out  of  them,  but  surely  in  that  case  it  would  have  ap- 
peared in  them  in  some  preparations.  She  explains  the  appear- 
ance of  the  reaction  in  the  blood  vessels  and  in  the  part  of  the 
parietal  cell  next  to  the  blood  vessel  (and  that  part  only)  by 
the  suggestion  that  under  certain  circumstances  the  acid  may  be 
secreted  by  the  parietal  cells  into  the  blood  stream  instead  of 
into  the  gland  lumen.  But  the  facts  she  reports  are  open  to  the 
other  interpretation  that  the  Prussian  blue,  or  the  salts  forming 
it,  may  have  been  excreted  from  the  blood  stream  into  the 
parietal  cell. 

Our  Own  Experiments. 

These  were  conducted  upon  rabbits,  cats,  dogs,  a  fowl,  a 
snapping  turtle,  and  several  skates.  Into  these  animals  we 
injected  solutions  of  sodium  ferrocyanide  (which  we  found  less 
toxic  than  potassium  ferrocyanide)  and  solutions  of  iron  and 
ammonium  citrate.  We  did  not  always  use  molecularly  balanced 
solutions,  because  we  found  that  the  two  salts  were  excreted 
with  very  different  degrees  of  rapidity  and  by  different  ways. 
The  injections  were  made  subcutaneously  or  intravenously.  In 
so  far  as  our  experiments  repeated  those  of  Miss  Fitzgerald,  they 
confirmed  the  results  reported  by  her  entirely,  but  we  obtained 
also  some  additional  results  which  have  a  very  important  bearing 
upon  the  conclusion  which  she  drew. 

By  these  experiments  we  sought  to  get  answers  to  the  following 
questions :  First,  is  the  Prussian  blue  precipitate  produced  in  any 
place  other  than  the  gastric  mucous  membrane? 


FORMATION   OF   HYDROCHLORIC   ACID   IN   THE   FOVEOL^.    23! 

Prussian  Blue  in  Places  Other  than  Gastric  Mucous  Membrane. 
—Into  the  ear  vein  of  a  rabbit  was  injected  13  c.c.  of  a  10  per  cent, 
solution  of  sodium  ferrocyanide,  and  into  the  other  ear  vein  10 
c.c.  of  a  25  per  cent,  solution  of  iron  and  ammonium  citrate;  both 
solutions  were  warm.  They  were  perfectly  fresh,  having  just 
been  made.  They  did  not  give  a  precipitate  of  Prussian  blue 
when  diluted  each  with  an  equal  quantity  of  water,  one  added  to 
the  other,  and  the  mixture  allowed  to  stand  in  a  warm  chamber 
at  37°  C.  for  28  hours.  The  rabbit  showed  some  toxic  effects 
during  the  injection,  rallied  temporarily,  but  died  in  20  minutes. 
The  stomach  was  opened  at  once.  It  was  full  of  fresh  food  in 
an  active  state  of  digestion.  Prussian  blue  appeared  in  all  parts 
of  it,  although  the  lesser  curvature  seemed  to  have  less  of  it 
than  the  rest  of  the  stomach.  Pieces  of  various  tissues  were  fixed 
in  absolute  alcohol.  The  urine  showed  both  salts  present  in 
abundance;  the  bile  contained  none  of  either.  The  saliva  con- 
tained ferric  citrate  but  no  ferrocyanide.  The  heart's  blood 
showed  no  blue  on  the  addition  of  acid  nor  on  the  addition  of 
acid  with  each  one  of  the  two  salts.  Therefore,  it  did  not  contain 
either  salt  in  any  quantity  appreciable  by  this  method.  Pararfine 
sections  were  cut  and  they  showed  the  Prussian  blue  reaction 
on  the  surface  of  the  stomach;  in  the  mucous  membrane  of  the 
stomach  appearing  in  the  blood  vessels,  in  the  lymphatic  spaces, 
in  the  epithelium  between  the  foveolse ;  in  the  connective  tissue  of 
the  muscularis  mucosse  and  of  the  tunica  muscularis  of  the 
stomach.  It  did  not  appear  in  the  parietal  cells  nor  in  any 
other  gland  cells,  nor  in  the  gland  lumen.  It  was  found  in  many 
other  tissues,  namely:  in  the  liver,  appearing  in  the  blood  vessels, 
in  the  endothelial  cells  of  Kupffer,  and  a  little  in  the  bile  capil- 
laries; in  the  spleen  appearing  in  the  blood  vessels;  in  the 
blood  vessels  of  the  heart  muscle.  These  were  the  only  places 
in  which  we  looked  for  it,  but  we  found  it  in  all  of  them. 

This  experiment  was  repeated,  using  smaller  quantities  of  the 
salts  injected  subcutaneously  on  two  successive  days  (a  total  of 
1.6  grammes  iron  and  ammonium  citrate  and  3.0  grammes  sodium 
ferrocyanide  well  diluted).  The  rabbit  was  killed  30  minutes 
after  the  second  injection;  the  tissues  were  fixed  in  formalin 
(neutralized  with  magnesium  carbonate).  The  Prussian  blue 


232  B.    C.    H.    HARVEY   AND    R.    R.    BENSLEY. 

reaction  was  found  in  all  places  in  which  it  was  found  in  the 
first  rabbit.  In  addition  it  was  found  in  the  villi  of  the  duodenum 
appearing  in  the  connective  tissue  near  the  free  end  of  the  villus, 
and  also  between  the  epithelial  cells  of  the  free  end  of  the  villus. 
The  blood  vessels  of  the  gastric  mucosa  contained  Prussian  blue, 
but  in  much  smaller  quantities  than  did  those  of  the  spleen  and 
liver.  The  parietal  cells  were  practically  free  of  it;  there  may 
have  been  a  little,  for  three  or  four  minute  particles  were  found 
in  the  section,  but  it  was  impossible  to  say  that  these  extremely 
minute  particles  were  not  on  the  surface  of  the  cell  instead  of 
within  it. 

From  these  two  experiments  it  is  evident  that  the  Prussian 
blue  is  precipitated  in  the  blood  stream  when  solutions  of  these 
salts  are  injected  into  it.  It  may  also  be  precipitated  or  absorbed 
in  various  places,  especially  in  the  endothelial  cells  of  Kupffer 
In  the  liver.  They  show  that  it  may  appear  on  the  surface  of 
the  stomach  when  it  is  not  in  the  parietal  cells;  that  it  appears 
in  the  blood  vessels  and  lymphatic  spaces  before  it  appears  in 
the  parietal  cells.  Its  appearance  between  the  cells  of  the  surface 
epithelium  between  adjacent  foveolse,  and  also  in  the  inter- 
glandular  blood  vessels  and  lymphatic  vessels  beneath  this 
epithelium  suggests  that  the  Prussian  blue  or  some  or  all  of  the 
salts  necessary  for  its  formation  may  pass  from  the  surface  into 
the  interglandular  lymphatics.  These  results  taken  in  conjunc- 
tion with  those  reported  by  Miss  Fitzgerald  in  her  rabbits  Nos. 
5  and  6  suggest  very  strongly  that  the  Prussian  blue  or  the 
constituents  forming  it  may  pass  from  the  blood  vessels  into 
the  parietal  cells  instead  of  in  the  reverse  direction  as  she  sup- 
posed. In  other  rabbits  we  have  frequently  seen  the  Prussian 
blue  in  the  blood  vessels  and  in  only  those  parts  of  a  few  parietal 
cells  lying  next  to  the  blood  vessels,  just  as  she  reports  in  her 
rabbits  5  and  6.  We  have  never  found  it  in  the  parietal  cells 
without  finding  it  also  in  the  blood  vessels,  but  in  these  two 
instances  we  found  it  in  the  blood  vessels  when  it  was  absent 
from  the  parietal  cells.  When  it  was  present  on  the  surface  of 
the  stomach  we  found  it  uniformly  present  also  between  the 
cells  of  the  interfoveolar  surface  epithelium  and  in  the  inter- 
glandular  lymphatic  vessels  and  blood  vessels.  We  have  fre- 


FORMATION   OF   HYDROCHLORIC   ACID   IN   THE   FOVEOUE.    233 

quently  found  it  similarly  in  the  tips  of  pyloric  and  duodenal 
villi,  between  the  epithelial  cells  and  in  the  subjacent  connective 
tissue.  Similar  results  were  obtained  in  a  fowl  which  had  been 
given  subcutaneously  13  c.c.  of  a  10  per  cent,  solution  of  sodium 
ferryocyanide  and  13  c.c.  of  a  25  per  cent,  solution  of  iron  and 
ammonium  citrate,  and  two  hours  later  intravenously  7  c.c. 
of  the  sodium  ferrocyanide  solution  and  4.5  c.c.  of  the  iron 
and  ammonium  citrate  solution;  both  solutions  were  fresh  and 
warm.  The  animal  stood  these  injections  very  well,  although 
they  had  some  toxic  effect.  The  foecal  passages  were  liquid  and 
contained  a  little  blue  precipitate  which  became  quite  heavy  and 
abundant  on  the  addition  of  hydrochloric  acid.  The  animal  was 
killed  in  one  hour  by  chloroform,  and  pieces  of  various  tissues 
were  fixed  in  absolute  alcohol.  When  the  proventriculus  was 
opened  a  blue  fluid  welled  out  of  the  openings  of  the  depressions 
which  contain  the  compound  glands;  no  blue  color  appeared  else- 
where in  this  organ.  Microscopic  examination  of  paraffine  sec- 
tions showed  Prussian  blue  on  the  surface  of  the  epithelium 
lining  the  necks  of  the  depressions  of  the  surface  epithelium 
into  which  the  glands  open  and  in  the  lumina  of  these  depressions. 
It  was  not  within  the  glands  themselves.  It  was  present  between 
the  epithelial  cells  of  the  region  where  the  depression  receiving 
the  secretion  of  the  glands  opened  on  to  the  surface  of  the  pro- 
ventriculus and  in  the  subjacent  blood  vessels  and  lymphatic 
spaces.  It  was  abundant  in  the  blood  vessels  and  lymphatic 
spaces  of  the  muscular  coat  of  the  stomach,  and  of  the  crop, 
and  of  the  heart  muscle,  and  of  the  muscular  stomach  (gizzard). 
It  appeared  also  in  the  epithelium  of  the  intestine  and  in  the 
connective  tissue  and  muscle  of  the  coecal  diverticula.  It  was 
abundant  in  the  liver  appearing  in  the  endothelial  cells  of  Kupffer 
and  in  the  blood  vessels.  It  was  absent  from  the  breast  muscle 
and  its  lymphatic  and  blood  vessels. 

These  results,  therefore,  answer  this  first  question  in  the 
affirmative.  The  Prussian  blue  reaction  appears  in  many  places 
besides  the  gastric  mucous  membrane.  In  most  of  them  it  seems 
probable  that  it  takes  place  without  the  help  of  an  acid.  And 
they  show  that,  if  its  occurrence  on  the  surface  of  the  gastric 
mucous  membrane  is  due  to  the  acid  formed  in  the  stomach  then 


234  B-    C.   H.   HARVEY   AND   R.    R.    BENSLEY. 

that  acid  is  present  on  the  surface  while  it  is  absent  from  parietal 
cells  and  from  the  gland  lumen. 

Effects  of  Injury  on  Amount  of  Prussian  Blue  Precipitate. — 
Second,  is  the  amount  of  Prussian  blue  precipitate  increased 
by  mechanical  injury  of  the  mucous  membrane?  A  small  kitten 
was  given  by  subcutaneous  injection  small  doses  of  a  mixture 
of  solutions  of  potassium  ferrocyanide  and  iron  and  ammonium 
citrate  in  molecular  proportions.  These  injections  were  given 
three  times  a  day  for  three  days.  On  the  third  day  the  abdomen 
was  opened,  a  small  piece  of  mucous  membrane  was  removed 
from  the  fundus  region  of  the  stomach,  the  wound  in  the  mucous 
membrane  was  sutured  with  silk  sutures  and  afterward  the 
other  coats  of  the  stomach  were  united  and  the  abdomen  was 
closed.  The  operation  was  done  with  aseptic  precautions.  Five 
hours  later  the  kitten  was  killed,  pieces  of  tissue  were  fixed  in 
alcohol  and  in  neutral  formalin  (freshly  distilled  over  potassium 
hydrate).  In  the  immediate  vicinity  of  the  suture  paramne 
sections  showed  that  the  Prussian  blue  reaction  occurred  in  great 
abundance  in  the  blood  vessels,  in  the  lymphatic  spaces  and  in 
the  parietal  cells.  These  preparations  showed  blue  in  a  very 
large  proportion  of  the  parietal  cells  and  even  in  those  at  the 
bottom  of  the  tubules.  It  was  especially  abundant  in  the 
parietal  cells  of  the  necks  of  the  glands.  Many  parietal  cells 
were  dead  and  thrown  off  into  the  gland  lumen;  in  every  instance 
these  dead  cells  were  filled  with  Prussian  blue.  In  those  cells 
which  were  still  in  their  normal  position  many  showed  the 
Prussian  blue  in  the  canaliculi  disposed  in  a  manner  very  similar 
to  that  which  Miss  Fitzgerald  has  shown  in  her  Plate  VII.,  Figs. 
6  and  8.  As  one  proceeded  in  the  study  of  these  sections  pro- 
gressively farther  from  the  site  of  injury  the  amount  of  Prussian 
blue  and  the  number  of  parietal  cells  showing  it  progressively 
decreased,  and  in  some  sections  of  mucous  membrane  taken 
from  parts  of  the  stomach  remote  from  the  site  of  operation  and 
apparently  in  a  healthy  condition,  the  parietal  cells  did  not  con- 
tain any  blue  at  all. 

In  another  cat  a  similar  operation  was  performed  on  the  gastric 
mucous  membrane  and  during  four  successive  days  following 
solutions  of  potassium  ferrocyanide  and  of  iron  and  ammonium 


FORMATION   OF  HYDROCHLORIC   ACID   IN   THE   FOVEOL^.    235 

citrate  in  equal  quantities  were  injected  subcutaneously.  On  the 
fourth  day  the  cat  died,  but  the  tissues  were  immediately  fixed 
in  neutral  formalin.  There  was  more  Prussian  blue  in  the 
immediate  neighborhood  of  the  sutures  than  in  other  parts  of 
the  mucous  membrane. 

These  results  answer  the  second  question  in  the  affirmative. 
And  they  show  that  dead  parietal  cells  show  more  Prussian 
blue  than  living  ones.  The  dead  ones  are  always  stained  by  it, 
whereas  most  of  the  living  ones  are  not.  This  suggests  that  the 
death  of  the  parietal  cell  or  a  lowering  of  its  vitality  may  permit 
or  facilitate  the  penetration  of  the  cell  by  substances  concerned 
in  the  Prussian  blue  reaction,  and  that  dead  cells  become  acid. 

Effect  of  Poisons  on  Amount  of  Prussian  Blue  Precipitate. — 
Third,  is  the  extent  of  the  Prussian  blue  reaction  increased  by  the 
injection  of  poisons  into  the  wall  of  the  stomach?  We  injected 
into  the  submucous  tissue  of  the  stomach  exposed  under  aseptic 
precautions,  solutions  of  phosphorus  in  olive  oil,  and  solutions  of 
moccasin  venom  in  distilled  water.  The  incisions  in  the  ab- 
dominal wall  were  closed  and  salts  of  sodium  ferrocyanide  and 
iron  and  ammonium  citrate  injected  subcutaneously  for  varying 
periods.  The  results  were  negative;  we  could  not  demonstrate 
any  increase  of  the  Prussian  blue  reaction  in  the  area  where  these 
poisons  were  injected. 

Effect  of  Restriction  of  Blood  Supply  on  Amount  of  Prussian 
Blue  Precipitate. — Fourth,  is  the  amount  of  Prussian  blue  in- 
creased in  areas  of  restricted  blood  supply? 

Arteries  and  veins  of  various  sizes  were  tied  on  the  stomachs 
of  different  rabbits  and  cats.  Solutions  of  sodium  ferrocyanide 
and  iron  and  ammonium  citrate  were  injected  subcutaneously 
during  several  days.  The  results  answered  this  question  in  the 
negative.  There  was  no  increase  of  Prussian  blue  in  the  areas  of 
restricted  blood  supply.  In  a  few  cases  ulcers  were  produced  and 
on  the  surface  of  these  there  was  always  a  considerable  deposit 
of  Prussian  blue.  The  cells  on  the  surface  of  the  ulcer  belonging 
to  various  parts  of  the  gastric  glands  and  presumably  dead  or 
dying  always  showed  a  considerable  deposit  of  Prussian  blue 
in  them. 

Precipitates  Do  Not  Back  into  Gland  Lumina  from  the  Surface. — 


236  B.    C.   H.    HARVEY   AND    R.    R.    BENSLEY. 

Fifth,  is  the  occasional  presence  of  Prussian  blue  in  the  gland 
lumen  and  in  the  canaliculi  of  the  parietal  cells  due  to  backing 
up  of  the  blue  precipitate  from  the  foveolae  or  from  the  surface  of 
the  mucous  membrane.? 

In  order  to  determine  this  question  many  experiments  were 
made  with  Prussian  blue,  with  carmine  and  with  India  ink. 
Hydrostatic  pressure,  dialysis,  and  positive  pressure  by  a  syringe 
piston  against  a  piece  of  gastric  mucous  membrane  tied  over  the 
end  of  the  syringe  were  employed.  The  results  were  all  nega- 
tive, thus  confirming  those  of  Lepine  ('72)- 

Prussian  Blue  Reaction  in  Animals  which  Have  no  Parietal 
Cells,  but  Secrete  Acid. — Sixth,  where  does  the  Prussian  blue 
reaction  occur  in  animals  which  have  no  parietal  cells  but  yet 
secrete  acid  ? 

The  experiment  above  reported  with  the  fowl  showed  that 
the  Prussian  blue  occurred  in  the  lumen  of  the  depression  receiv- 
ing the  secretion  of  the  compound  glands  of  the  proventriculus. 
It  was  not  found  in  the  gland  cells.  It  appeared  between  the 
cells  of  the  surface  epithelium  immediately  adjacent  to  the 
opening  of  the  depressions.  As  above  stated,  it  occurred  also 
in  many  other  places. 

A  snapping  turtle  was  given  one  half  gram  each  of  sodium 
ferrocyanide  and  iron  and  ammonium  citrate  in  dilute  solution 
three  times  daily  during  four  days;  the  solutions  werte  given 
separately  and  subcutaneously  in  the  inguinal  region.  Two 
small  fish  were  shoved  into  the  stomach  with  a  glass  rod  and  were 
found  there  later  partially  digested.  On  the  fourth  day  the 
animal  was  killed  and  the  stomach  examined.  A  deposit  of 
Prussian  blue  was  found  in  every  cell  of  the  somewhat  coarse 
foveolae  of  the  gastric  gland.  Iri  these  cells  it  occupied  a  definite 
position,  the  same  in  each  cell.  Each  cell  contained  a  mucous 
plug  which  occupied  the  half  of  the  cell  next  the  lumen,  under 
it  a  small  spherical  mass  of  Prussian  blue,  and  under  that  the 
nucleus.  The  mass  of  Prussian  blue  was  nearly  as  large  as  the 
nucleus.  Sections  through  these  foveolae  stained  with  paracar- 
mine  or  mucicarmine  presented  a  very  pretty  appearance 
because  of  the  extreme  regularity  of  the  position  of  the  nuclei, 
the  little  masses  of  Prussian  blue  and  the  mucous  plugs.  There 


FORMATION   OF   HYDROCHLORIC   ACID   IN   THE   FOVEOL^.    237 

was  more  Prussian  blue  in  the  cells  of  the  foveolar  epithelium 
then  anywhere  flse.  The  amount  in  the  cells  of  the  necks  of  the 
gland  was  less  and  decreased  toward  the  bottom  of  the  gland 
tubule,  although  it  appeared  in  nearly  all  the  cells  of  the  gland. 
It  was  abundant  in  the  interglandular  connective  tissue,  being 
in  the  blood  and  lymph  vessels. 

Skates  experimented  upon  in  the  same  way  showed  Prussian 
blue  in  the  blood  vessels  and  lymphatics  of  the  wall  of  the 
pharynx  and  stomach.  The  glands  of  the  gastric  mucous  mem- 
brane showed  the  reaction  in  small  quantities  in  the  gland  lumina 
and  in  a  few  of  the  cells.  The  cells  showing  the  reaction  were 
more  numerous  in  the  part  of  the  gland  near  the  free  surface 
than  in  its  deeper  portions.  The  surface  epithelium  of  the 
stomach  and  of  the  intestines  two  inches  beyond  the  pylorus 
and  of  the  large  intestine  contained  small  quantities  of  Prussian 
blue. 

Summary  of  the  Results  Obtained  by  the  Prussian  Blue  Reaction 
and  their  Significance. 

From  the  facts  reported  by  Miss  Fitzgerald  and  those  which 
our  own  experiments  have  added,  it  seems  clear  that  the  occur- 
rence of  the  Prussian  blue  reaction  does  not  necessarily  indicate 
the  formation  of  free  mineral  acid  under  normal  conditions 
in  the  places  in  which  it  is  found.  It  appears  in  many  places 
where  it  could  hardly  have  been  due  to  the  presence  of  acid — 
in  the  blood,  lymph,  liver,  spleen,  intestine,  heart  muscle,  etc. 
In  these  places  its  presence  must  be  due  to  something  else, 

It  may  be  due  to  fatty  acids,  which  bring  about  the  precipi- 
tation of  Prussian  blue  from  solutions  of  the  salts  used  in  these 
experiments  in  proportion  to  the  amount  of  the  acid  present. 
It  may  be  due  to  the  withdrawal  of  the  ammonium  citrate  by 
more  rapid  diffusion  or  by  the  involvement  of  the  ammonium  in 
the  metabolic  processes  of  the  tissues.  Nencki  and  Pawlow  ('96) 
have  shown  that  the  gastric  mucous  membrane  normally  con- 
tains an  extraordinarily  large  amount  of  ammonia.  Ferric  citrate 
and  sodium  ferrocyanide  solutions  give  when  mixed  an  imme- 
diate precipitate  of  Prussian  blue  even  in  the  absence  of  any  acid. 
It  may  be  due  to  the  death  or  reduced  vitality  of  cells,  permitting 


238  B.    C.   H.   HARVEY   AND    R.    R.    BENSLEY. 

the  entrance  of  salts  which  would  not  have  entered  living  or 
healthy  cells.  It  may  be  that  the  cells  which  show  the  reaction 
are  just  those  which  are  poisoned  by  the  salts  used.  The  iron 
and  ammonium  citrate  solution  .injected  repeatedly  during 
several  days  caused  the  death  of  one  kitten.  Therefore  this 
salt  has  a  serious  toxic  action.  It  may  be  that  the  interaction 
chemically  of  these  two  salts  with  the  contents  of  certain  cells 
may  sometimes  permit  the  liberation  of  an  acid  in  them,  when 
no  acid  would  have  been  produced  in  the  absence  of  the  com- 
plex chemical  state  which  exists  when  they  are  present.  It  may 
have  been  absorbed  from  the  surface  of  the  mucous  membrane. 
Its  presence  between  the  epithelial  cells  and  in  underlying  lym- 
phatic vessels  of  the  stomach  and  intestine  suggest  this  possi- 
bility. 

Since  the  Prussian  blue  may  be  precipitated  in  so  many  places, 
the  fact  that  it  is  sometimes  precipitated  in  the  canaliculi  of  a 
few  parietal  cells  in  a  relatively  small  part  of  the  stomach  per- 
haps in  an  abnormal  condition  at  the  time  does  not  necessarily 
prove  that  free  hydrochloric  acid  is  formed  under  normal  con- 
ditions in  the  parietal  cells  of  the  stomach  as  a  whole.  The 
failure  to  get  any  reaction  in  the  stomach  in  some  experiments, 
the  small  number  of  parietal  cells  in  which  it  ever  appears,  its 
occurrence  in  other  tissues  and  in  the  blood  vessels  and  lymph 
vessels  before  it  appears  in  the  parietal  cells  at  all,  the  fact  that 
several  factors  other  than  the  presence  of  free  mineral  acid  may 
cause  the  precipitate  to  form, — all  these  things  show  that  it 
would  not  be  right  to  conclude  from  the  evidence  which  the 
Prussian  blue  reaction  affords  that  free  hydrochloric  acid  is 
formed  in  the  paiietal  cells.  Much  less  could  one  reach  this 
conclusion  from  any  other  evidence  that  has  been  adduced,  for 
all  other  evidence  is  much  less  definite  than  this.  And  this 
failure  to  show  clearly  that  free  hydrochloric  acid  is  formed  in 
the  parietal  cells  becomes  quite  clear  when  it  appears,  as  we  shall 
show  in  the  following  part  of  this  paper,  that  the  contents  of  the 
canaliculi  of  these  cells  are  alkaline  and  those  of  the  gland  lumina 
are  not  acid  when  free  acid  is  being  produced  by  the  mucous 
membrane. 


FORMATION   OF  HYDROCHLORIC  ACID   IN   THE    FOVEOL/E.    239 

EXPERIMENTS  WITH  INDICATORS:   THE  REACTION  OF  THE  SECRE- 
TION WITHIN  THE  LUMEN  OF  THE  ACTIVE  FUNDUS  GLAND, 
AND  WITHIN  THE  INTRACELLULAR  CANALICULI 
OF  THE  PARIETAL  CELLS. 

It  is  obvious  from  the  observations  of  Fitzgerald  and  ourselves 
reported  in  the  preceding  section  that  the  Prussian  blue  reaction 
is  not  a  trustworthy  indication  of  the  place  of  formation  of  the 
hydrochloric  acid  of  the  gastric  juice,  and  that  we  must  look 
to  other  methods  for  a  solution  of  this  problem.  One  naturally 
turns  to  the  chemical  indicators  for  this  purpose.  The  results 
obtained  by  means  of  these  substances  by  previous  investigators 
who  have  employed  them  have  been  uniformly  unsatisfactory 
and  unconvincing.  The  most  definite  results  obtained  by  these 
methods  are  those  reported  by  Frankel  ('91)  who  used  neutral 
sodium  rosanilinsulphonate  as  an  indicator,  and  by  Edinger 
('79),  who  employed  a  solution  of  sodium  alizarin. 

Rosanilin  sulphonic  acid  possesses  the  property  of  forming 
with  sodium  hydroxide  acid  salts  which  form  red  solutions  (acid 
fuchsin),  and  neutral  salts  whose  solutions  are  colorless.  The 
addition  of  small  quantities  of  acid  to  solutions  of  the  neutral 
salt  results  in  the  production  of  the  red  colored  acid  salt.  Ac- 
cordingly, Frankel  injected  into  the  jugular  veins  of  dogs  50- 
100  c.c.  of  a  5  per  cent,  solution  of  the  neutral  sodium  rosanilin 
sulphonate.  As  a  result  of  this  proceeding  he  found  the  entire 
mucous  membrane  of  the  stomach,  including  the  pyloric  mucous 
membrane,  stained  brilliant  red.  Teasing  portions  of  the  mucous 
membrane  in  distilled  water  he  found  that  both  parietal  and  chief 
cells  were  stained  in  the  fundus  glands,  and  that  the  cells  of  the 
pyloric  glands  were  also  stained,  while  the  cylindrical  cells  of 
the  surface  were  unstained.  He  could  see  no  difference  in  the 
intensity  of  the  stain  in  the  two  types  of  cells.  The  same  experi- 
ment was  also  performed  on  rabbits,  but  in  this  case  he  found 
that  the  color  was  not  uniformly  distributed  throughout  the 
mucous  membrane,  and  the  pyloric  mucosa  showed  only  a  few 
slightly  red  spots. 

From  these  experiments  Frankel  concludes:  that  the  mucous 
membrane  of  the  stomach  has  an  acid  reaction;  that  the  acid  is 
formed  in  the  parenchyma  cells;  and  that  it  can  always  be 


240  B.    C.   H.   HARVEY   AND   R.    R.    BENSLEY. 

demonstrated  in  them.  Regarding  the  acid  reaction  obtained 
by  this  method  in  the  pyloric  region  he  does  not  venture  an 
interpretation. 

The  results  obtained  by  Edinger  by  means  of  the  sodium 
alizarin  reaction  were  similar  to  those  of  Frankel.  Sodium  ali- 
zarin, as  Edinger  pointed  out  is,  in  neutral  solutions,  of  a  deep 
purple  red  color,  while  the  addition  of  an  acid  results  in  the  pre- 
cipitation of  the  alizarin  as  a  flocculent  yellow  precipitate. 
Edinger  prepared  the  solution  by  adding  alizarin  in  excess  to  a 
10  per  cent,  solution  of  sodium  hydroxide.  Then  the  solution 
was  filtered.  25-100  c.c.  of  this  solution  were  injected  into  the 
jugular  veins  of  rabbits  and  dogs.  In  a  rabbit  he  found  after 
this  injection  the  stomach  spotted  red-violet  and  yellow,  the 
latter  being  more  general  in  the  region  of  the  greater  curvature, 
though  the  pyloric  mucous  membrane  was  also  yellowr.  He 
concludes  that  the  glands  of  the  rabbit's  stomach  are  not  all  in 
activity  at  the  same  time,  and  that  both  the  fundus  and  pyloric 
mucous  membranes  react  acid.  In  dogs,  after  similar  treatment, 
the  whole  mucous  membrane  of  the  stomach,  including  that  of 
the  pyloric  region,  was  yellow.  Sections  of  the  mucous  mem- 
brane showed  that  the  yellow  color  was  to  be  found  at  all  levels, 
but  the  intensity  of  the  stain  was  too  slight  to  permit  of  the 
recognition  of  the  stain  in  particular  cells.  The  pancreas  also 
gave  an  acid  reaction. 

Experiments  with  tropeolin,  congo  red,  litmus,  phenol- 
phthalein,  and  other  indicators  in  common  use,  have  been  with- 
out result  or,  at  the  most,  have  only  indicated  what  was  known 
from  examination  of  the  secretion,  namely,  that  the  contents  of 
the  stomach  were  acid. 

It  is  obvious  from  the  foregoing  statements  that  the  results  of 
the  experiments  of  Edinger  and  Frankel  with  sodium  alizarin 
and  sodium  rosanilin  sulphonate  were  not  in  accord  with  what 
had  been  previously  determined  concerning  the  place  of  forma- 
tion of  hydrochloric  acid  in  the  stomach,  inasmuch  as  they 
indicated  the  formation  of  acid  in  the  pyloric  mucous  membrane 
which  had  previously  been  shown  by  Heidenhain  ('70)  and 
Klemensiewicz  ('75)  to  secrete  an  alkaline  fluid.  Moreover, 
neither  of  these  experiments  gave  any  clear  indication  of  the 
.Source  of  the  hydrochloric  acid. 


FORMATION   OF   HYDROCHLORIC   ACID   IN   THE   FOVEOL^.     24! 

It  is  also  clear  that  in  order  to  solve  this  problem  by  the  use 
of  a  chemical  indicator  the  substance  employed,  in  addition  to 
being  an  indicator  of  acidity  or  alkalinity,  must  have  the  prop- 
erties of  a  vital  stain,  that  is  to  say,  the  cells  of  the  gastric  glands 
must  be  freely  permeable  to  it  and  it  must  have  a  special  affinity 
for  constituents  of  the  gastric  secretion  in  the  glands,  or  the 
distribution  coefficient  must  favor  its  concentration  in  this 
secretion  in  sufficient  amounts  to  give  a  distinct  color  reaction. 
These  conditions  we  have  found  to  be  fulfilled  by  neutral  red 
and  by  a  number  of  dyes  belonging  to  the  naphtol  blue  series 
including  nile  blue,  and  the  various  cyanamins  discovered  by 
Witt  ('90). 

Our  first  successful  experiments  in  staining  specifically  the 
secretion  in  the  parietal  cells  and  in  the  lumina  of  the  gastric 
glands  were  obtained  with  Griibler's  napthalin  blue  R  crystals 
(a  trade  name  for  naphtol  blue).  Solutions  of  this  dye  in  normal 
salt  solution,  injected  into  the  blood  vessels  of  the  recently  killed 
animal,  were  found  to  stain  the  secretion  in  the  canaliculi  of  the 
parietal  cells  and  in  the  lumina  of  the  gland  tubules  of  the  fundus 
region  a  distinct  red  color,  while  the  cells  of  the  foveola  and  the 
mucus  on  the  free  surface  were  stained  a  deep  blue. 

Tests  of  the  solution  of  naphtalin  blue  afforded  no  explana- 
tion of  this  result,  inasmuch  as  addition  of  acid  produced  no 
change  in  the  color  of  the  solution,  and  addition  of  sodium 
hydrate  gave  a  green  color.  Accordingly,  it  seemed  probable 
that  the  reaction  observed  was  due  either  to  another  dye  present 
in  the  naphtalin  blue  as  an  impurity,  or  to  a  new  dye  synthesized 
during  the  process  of  staining. 

After  consideration  of  the  commercial  process  for  the  manu- 
facture of  the  naphtol  blues  it  seemed  probable,  in  view  of  the 
fact  that  dimethylparaphenylendiamin  is  a  biproduct  of  the 
synthesis  of  naphtol  blue  from  nitrosodimethylanilin  and  B 
naphtol,  that  the  dye  on  which  this  reaction  depended  would 
prove  to  be  cyanamin,  which,  according  to  Witt,  is  formed  when 
the  mixture  of  naphtol  blue  and  dimethylparaphenylendiamin 
resulting  from  the  synthesis  above  mentioned  is  boiled  for  a  time 
with  an  alcoholic  solution  of  potassium  hydroxide.  Accordingly, 
cyanamin  chloride  was  prepared  by  the  process  described  by 
Witt,  and  its  solutions  tested  on  the  gastric  mucous  membrane. 


242  B.    C.    H.   HARVEY   AND    R.    R.    BENSLEY. 

Cyanamin,  according  to  Witt,  possesses  two  basic  groups,  one 
molecule  of  the  base  combining  with  two  molecules  of  hydro- 
chloric acid  to  form  a  bichloride  which  is  soluble  in  water  with  a 
deep  blue  color.  On  dilution  of  this  solution  the  compound  is 
broken  up  into  a  monochloride  insoluble  in  water,  which  deposits 
as  a  reddish  violet  precipitate,  and  hydrochloric  acid,  which 
remains  in  solution.  On  the  addition  of  alkalies  the  solution 
changes  to  a  red  color  and  after  a  short  Yime  the  base  settles  out 
as  a  red  flocculent  precipitate. 

On  account  of  the  formation  of  a  monochloride  intermediate 
in  color  between  the  red  base  and  the  blue  bichloride  it  is  appar- 
ent that  as  an  indicator  of  reaction  cyanamin  does  not  approach  in 
delicacy  of  response  the  more  commonly  used  chemical  indicators. 
But  when  we  consider  the  relatively  high  content  of  hydrochloric 
acid  in  the  gastric  juice  (as  high  as  .5822  per  cent,  according 
to  Rosemann  ('07))  this  is  of  little  importance,  for  we  have 
found  that  in  dilute  solutions  of  the  dye  a  concentration  of  .0009 
per  cent,  of  hydrochloric  acid,  or  approximately  1/600  of  the 
concentration  in  the  gastric  juice,  is  sufficient  to  abolish  all  trace 
of  red  color.  Furthermore,  if  Pawlow's  idea  is  true  that  the 
native  secretion  has  a  constant  acidity,  and  that  the  variations  in 
acidity  of  the  secretion  from  a  gastric  fistula  are  due  to  different 
degrees  of  neutralisation  by  the  alkaline  mucous  secretion  of 
the  surface  epithelium,  then  we  might  expect  a  maximum  acidity 
in  the  gland  lumen  assuming  that  the  hydrochloric  acid  is  secreted 
as  such  by  the  cells.  It  follows  therefore  that  if  cyanamin  stains 
the  gastric  secretion  in  the  glands  it  will  stain  it  blue  wherever 
the  acid  is  produced. 

The  method  of  applying  the  cyanamin  is  as  follows:  A  fresh 
concentrated  solution  of  the  bichloride  in  normal  sodium  chloride 
solution  is  prepared;  the  animal  is  killed  by  a  blow  on  the  head, 
or  by  bleeding  from  the  carotid,  and  the  stomach  exposed  as 
rapidly  as  possible;  a  small  piece  of  the  mucous  membrane  is 
cut  out  with  scissors,  rinsed  in  normal  salt  solution,  and  placed 
in  the  solution  of  the  dye.  A  few  minutes'  immersion  suffices 
to  accomplish  the  staining.  When  this  is  complete  the  piece 
of  mucous  membrane  is  placed  on  a  slide  with  the  mucous  surface 
downwards  and  observed  with  a  low  power  of  the  microscope. 


FORMATION   OF  HYDROCHLORIC  ACID   IN   THE   FOVEOL^.      243 

If  the  staining  has  progressed  far  enough  the  edge  of  the  prepara- 
tion may  be  teased  with  needles  and  the  superficial  glands  which 
alone  are  stained  so  isolated,  when  a  cover  glass  is  applied  and 
the  preparation  studied  by  high  power  objectives.  For  these 
experiments  we  have  used  rabbits,  guinea  pigs,  cats,  and  dogs. 

In  such  preparations  certain  cells  scattered  throughout  the 
glands  promptly  stain  blue,  the  blue  color  affecting  not  only 
the  protoplasm  but  the  nucleus.  These  belong  to  both  classes 
of  cells  constituting  the  glands  and  are  interpreted  by  us  as  dead 
cells.  In  addition  the  small  cells,  first  described  by  R.  Heiden- 
hain,  which  occur  in  small  numbers  scattered  among  the  other 
epithelial  cells  of  the  gland,  and  the  nature  of  which  is  still 
obscure,  stain  blue,  but  in  this  case  the  blue  stain  is  confined  to 
the  granules  with  which  the  protoplasm  of  these  cells  is  studded, 
the  nucleus  remaining  unstained.  Certain  glands  on  the  very 
edge  of  the  preparation  may  stain  bluish  red,  these  being  for 
the  most  part  glands  which  have  been  actually  injured  in  making 
the  preparations. 

In  the  uninjured  glands  reached  by  the  dye,  on  the  contrary, 
a  uniform  and  characteristic  reaction  is  obtained.  With  the 
exception  of  the  dead  cells  and  the  small  cells  of  Heidenhain  men- 
tioned above,  the  dye  is  entirely  confined  to  the  secretion  in  the 
lumina  of  the  glands  and  their  various  diverticula,  including  the 
whole  basketwork  of  canaliculi  in  the  parietal  cells — all  of  which 
was  intensely  stained.  Moreover,  in  no  place  in  this  system  of 
gland  tubules  below  the  level  of  the  gastric  foveolae  was  the  blue 
color  of  the  acid  solutions  of  the  dye  obtained.  On  the  contrary 
the  secretion  contained  in  the  canaliculi  of  the  parietal  cells  was 
a  distinct  red  like  that  displayed  by  the  dye  in  alkaline  solutions, 
while  the  secretion  in  the  lumen  of  the  gland  was  a  bluish  red. 
The  short  canaliculi  connecting  the  parietal  cell  system  of  intra- 
cellular  channels  with  the  main  lumen  of  the  gland  showed  a 
color  shading  from  the  red  of  the  content  of  the  latter  to  the 
bluish  red  of  the  contents  of  the  gland  lumen.  At  the  level  of 
the  bottoms  of  the  foveolae  the  color  of  the  secretion  changed 
rapidly  to  the  pure  blue  of  the  acid  solutions  of  cyanamin,  and 
the  cylindrical  cells  of  the  surface  and  of  the  foveolae  stained  the 
acid  color  also. 


244  B-    C-    H-    HARVEY   AND    R.    R.    BENSLEY. 

Inasmuch  as  the  results  just  described  indicated  that  in  no 
part  of  the  gland  system  below  the  foveolar  level  did  the  secretion 
of  the  gastric  gland  cells  have  an  acid  reaction,  and  that  the 
secretory  contents  of  the  parietal  cells  were  even  alkaline  in 
reaction  it  was  important  to  test  the  behavior  in  different  states 
of  physiological  activity  of  these  glands  towards  solutions  of 
the  dye.  These  experiments  were  performed  on  dogs,  animals 
being  kept  without  food  for  twenty-four  hours,  and  compared, 
as  regards  the  reaction  with  cyanamin  chloride,  with  other 
animals  at  different  intervals  after  feeding.  These  experiments 
showed  that  the  resting  gastric  glands  gave  no  reaction  with 
cyanamin,  while  glands  taken  from  active  stomachs  fifteen  or 
more  minutes  after  secretion  gave  the  pronounced  and  char- 
acteristic reaction  described  above.  Accordingly,  the  alkaline 
reaction  of  the  contents  of  the  canaliculi  of  the  parietal  cells,  and 
the  non-acid  reaction  of  the  contents  of  the  lumen  of  the  gland 
proper,  are  not  the  reactions  of  resting  glands,  but  only  of  active 
glands  from  a  stomach  which  is  forming  an  acid  secretion. 

The  amount  of  cyanamin  chloride  at  our  disposal  did  not  permit 
our  testing  its  action  on  the  stomach  when  injected  into  the  living 
animal  intravenously,  or  by  injection  immediately  after  death 
of  solutions  through  the  blood  vessels.  Naphtol  blue,  however, 
apparently  owes  its  properties  in  this  connection  to  admixture 
of  cyanamin,  or  to  synthesis  of  the  latter  during  the  process  of 
staining,  for  the  reaction  which  it  gives  is  exactly  that  of  the 
pure  cyanamin  solutions,  and  we  have  been  able  to  separate  from 
the  commercial  zinc  naphtol  blue  double  chloride  small  quantities 
of  cyanamin.  Naphtol  blue  dissolved  in  normal  salt  solution 
injected  from  the  aorta  in  a  rabbit  killed  shortly  after  feeding 
will  produce  this  reaction  in  every  gland  of  the  fundus  region  of 
the  stomach.  It  is  difficult,  however,  by  this  method,  to  secure 
a  staining  of  the  entire  gland,  for  reasons  which  a  consideration 
of  the  blood  supply  of  the  mucous  membrane  will  make  apparent. 
The  bases  of  the  glands  stain  well,  but  it  is  difficult  to  secure  a 
staining  of  the  upper  portions  of  the  glands.  Preparations  made 
in  this  way  give,  however,  the  most  remarkable  demonstration 
of  the  canalicular  system  of  the  glands  that  we  have  ever  seen, 
resembling  except  for  the  color  a  perfect  silver  chromate  impreg- 


FORMATION   OF   HYDROCHLORIC   ACID   IN   THE   FOVEOL^.     245 

nation  of  this  system.     We  have  also  obtained  a  feeble  reaction 
by  means  of  intravenous  injection  of  solutions  of  naphtol  blue. 

CONFIRMATORY  TESTS. 

In  view  of  the  results  obtained  with  cyanamin  chloride  it 
seemed  probable  that  other  dyes  closely  related  to  this  substance 
would  give  similar  reactions,  and  accordingly  we  prepared  by 
acting  on  naphtol  blue  with  anilin,  according  to  the  method 
described  by  Nietzki  and  Bossi  ('92),  the  closely  related  dye 
named  by  the  former  phenylated  nile  blue.  Solutions  of  this 
dye  gave  by  far  the  most  striking  results  obtained  inasmuch  as 
the  intensely  red  base  was  precipitated  in  the  canaliculi  of  the 
parietal  cell,  while  the  secretion  in  the  gland  lumen  was  stained 
a  bluish  red  color.  Similar  results  were  obtained  with  solutions 
of  nile  blue  sulphate,  but  a  less  pronounced  reaction  was  obtained, 
the  content  of  the  parietal  cells  staining  in  this  case  bluish  red, 
that  of  the  lumina  of  the  glands  blue. 

Neutral  red,  which  has  been  highly  commended  by  Ehrlich 
as  an  indicator  for  biological  studies,  next  suggested  itself  in  this 
connection  for  we  had  long  known  that  it  stained  the  secretion 
in  the  gastric  glands  and  in  the .  parietal  cells.  This  dye  may 
be  used  like  cyanamin  by  immersing  the  fresh  mucous  membrane 
in  a  i  in  10,000  solution  in  normal  salt  solution  or  by  injecting 
such  a  solution  of  the  dye  through  the  blood  vessels.  In  neutral 
solutions  neutral  red  possesses  a  reddish  color  with  a  suggestion 
of  orange.  Alkaline  solutions  precipitate  the  base  in  the  form 
of  a  yellow  precipitate  while  acid  solutions  produce  a  crimson 
color.  This  dye  therefore  is  capable  of  indicating  either  acidity, 
alkalinity  or  neutrality.  In  preparations  made  as  indicated 
above  of  the  fresh  actively  secreting  fundus  mucous  membrane 
of  the  stomach  neutral  red  promptly  stains  the  secretion  in  the 
canaliculi  of  the  parietal  cells  and  in  the  main  lumen  of  the 
gland.  In  the  parietal  cells  the  color  assumed  is  the  unmistakable 
yellow  of  the  free  base,  in  the  lumen  of  the  gland  the  color 
approaches  more  closely  to  the  neutral  tint,  while  the  short 
diverticula  of  the  lumina  which  connect  the  parietal  cell  with  the 
lumen  are  of  an  intermediate  tint.  The  whole  system,  however, 
is  without  question  on  the  alkaline  side  of  the  reaction  with 


246  B.    C.    H.    HARVEY   AND    R.    R.    BENSLEY. 

neutral  red.  At  the  bottom  of  the  foveolse  the  alkaline  reaction 
gives  way  to  the  crimson  acid  color  which  is  exhibited  by  the  whole 
foveola  and  by  the  surface.  The  foveolar  epithelium  also  stains 
the  crimson  acid  tint.  Neutral  red  stains  the  dead  cells  deep 
red,  and  also  the  granules  of  the  small  cells  of  Heidenhain  referred 
to  above. 

Thus  the  consistent  results  of  four  separate  methods  show 
that  the  hydrochloric  acid  is  not  free  as  such  in  the  gland,  and 
that  the  contents  of  the  canaliculi  of  the  parietal  cell  contrary 
to  expectation  are  alkaline  in  reaction.  The  question  naturally 
arises,  then,  wrhere  is  the  acid  of  the  gastric  juice  formed  and  what 
are  the  factors  concerned  in  its  formation?  Without  doubt, 
our  reactions  with  the  dyes  of  the  cyanamin  series  indicate  that 
the  hydrochloric  acid  of  the  gastric  juice  is  set  free  in  the  foveola, 
possibly  also  on  the  free  surface  of  the  mucous  membrane.  As 
to  the  source  of  the  chlorine  concerned  in  the  formation  of  hydro- 
chloric acid  of  the  stomach  the  experiments  of  Greenwood  ('85), 
Macallum  ('08),  and  Fitzgerald  ('10)  seem  to  be  conclusive. 
Greenwood  showed  that  in  preparations  of  the  mucous  membrane 
of  the  stomach  made  with  silver  nitrate,  and  then  reduced  in  the 
light,  the  parietal  cells  stained  much  more  strongly  with  the 
silver  deposit  than  the  other  epithelial  elements.  In  his  studies 
of  the  silver  reaction  for  chlorides  Macallum  showed  that  only 
chlorides,  phosphates,  and  carbonates,  of  silver  gave  this  reduc- 
tion reaction,  and  devised  a  method  by  means  of  which  the 
phosphates  and  carbonates  could  be  excluded  and  only  chlorides 
exhibited.  This  method  consisted  in  using  for  the  reaction  a 
solution  of  silver  nitrate  containing  nitric  acid  in  which  the 
phosphates  and  carbonate  of  silver  are  soluble.  By  this  means 
he  demonstrated  that  the  parietal  cells  of  the  stomach  were  rich 
in  chlorides.  This  result  has  recently  been  confirmed  by  Miss 
Fitzgerald,  who  found  that  the  reaction  was  obtained  not  only  in 
the  body  of  the  parietal  cell  but  also  in  the  intracellular  channels. 

This  being  the  case,  in  view  of  the  fact  that  the  secretion  of 
the  parietal  cells  is  alkaline  while  in  the  cells  themselves,  and 
that  the  secretion  of  the  whole  gland  while  contained  in  the  gland 
lumen  is  very  nearly  neutral  as  shown  by  the  neutral  red  and 
cyanamin  reactions,  it  seems  probable  that  the  chlorine  is 


FORMATION    OF   HYDROCHLORIC   ACID   IN   THE   FOVEOL^.     247 

secreted  by  the  parietal  cells  in  the  form  of  a  chloride  of  an 
organic  base,  and  that  the  hydrochloric  acid  is  only  set  free 
after  this  secretion  is  poured  out  of  the  gland  into  the  foveola. 
As  to  the  nature  of  this  base,  there  are  some  facts  which  suggest 
the  probability  that  it  is  protein  in  nature.  Stohr's  ('82)  descrip- 
tion of  the  parietal  cells  in  man  indicates  clearly  that  he  perceived 
a  coagulated  substance  in  the  canals  which  connect  these  cells 
with  the  lumen  of  the  gland,  and  Revell  has  succeeded  in  staining 
the  content  of  the  intracellular  canals  of  the  parietal  cells  with 
carmin  solutions  in  material  fixed  in  an  alcohol  bichromate 
sublimate  mixture. 

THE  CONSISTENCE  OF  THE  SECRETION  IN  THE  GLAND  LUMEN. 

The  fact  that  we  were  able  to  stain  the  secretion  of  the  gastric 
glands  while  still  contained  in  the  gland  has  enabled  us  to  study 
certain  properties  of  this  secretion.  In  the  actively  secreting 
rabbit  stomach  the  lumen  of  the  gland  is  widened  by  the  accumu- 
lation of  the  secretion,  and,  by  pressure  on  the  cover  glass,  or  by 
teasing,  it  is  possible  to  expel  the  secretion  from  the  gland,  or  to 
liberate  it  in  the  salt  solution  used  for  mounting  and  thus  to 
learn  something  about  the  change  in  concentration  of  the  secre- 
tion which  takes  place  as  it  proceeds  towards  the  surface  of  the 
mucous  membrane.  The  assumption  which  is  generally  made 
that  the  secretion  is  formed  by  the  glands  in  the  same  concentra- 
tion as  it  presents  when  it  emerges  from  the  openings  of  the 
foveolae,  would  lead  one  to  suppose  that  the  secretion  in  the 
gland  would  be  a  limpid  solution,  which  would  flow  easily  from 
the  gland,  and  would  mix  readily  with  salt  solution.  This, 
however,  proved  not  to  be  the  case.  When  water  from  the  sur- 
rounding salt  solution  enters  the  gland  lumen  the  column  of 
secretion  breaks  up  into  round  droplets  which  maintain  their 
individuality  for  several  minutes.  Similarly,  when  secretion  is 
expressed  from  the  gland  lumen  into  the  surrounding  solution  it 
collects  around  the  mouth  of  the  gland  in  large  spherical  droplets 
which  slowly  dissolve,  the  red  reaction  also  at  the  same  time 
slowly  changing  to  the  blue  acid  reaction,  if  the  secretion  has  been 
stained  with  cyanamin.  From  these  observations  we  are  obliged 
to  conclude  that  the  secretion  formed  in  the  gland  possesses  a 


248  B.    C.    H.    HARVEY   AND    R.    R.    BENSLEY. 

relatively  high  content  of  solids,  and  that  the  bulk  of  the  water 
found  in  the  gastric  secretion  is  added  at  the  level  of  the  glandular 

foveolse. 

OTHER   CONSIDERATIONS. 

Since  it  is  apparent  that  the  contents  of  the  gastric  glands  proper 
when  in  a  state  of  normal  activity  are  not  acid  in  reaction,  and 
may  even  be  alkaline,  it  follows  that  the  ferment  of  the  same 
secretion  in  the  gland  lumen  is  probably  not  in  an  active  form, 
since,  as  is  well  known,  pepsin  is  destroyed  by  alkalies  while 
pepsinogen  is  not  affected.  Hence,  the  failure  of  the  secretion 
to  attack  the  cells  themselves  requires  no  further  explanation  in 
the  case  of  the  gastric  glands  than  in  the  case  of  the  pancreas, 
since  in  neither  case  does  the  activated  ferment  come  in  immediate 
contact  with  the  parenchyma  cells.  This  being  the  case  it  is 
pertinent  to  enquire  whether  under  any  conditions  the  secretion 
within  the  gland  may  become  acid  in  reaction,  for,  in  this  event, 
it  is  probable  that  the  ferment  would  be  activated  and  as  happens 
under  similar  conditions  in  the  pancreas  the  adjacent  parenchyma 
cells  would  be  attacked.  This  possibility  is  suggested  by  certain 
results  obtained  with  the  Claude  Bernard  reaction,  where  a 
reaction  was  obtained  in  the  neighborhood  of  recent  injuries  to 
the  mucous  membrane  far  down  the  lumen  of  the  gland,  though 
the  rest  of  the  mucous  membrane  showed  no  reaction  in  the 
glands.  We  have  as  yet  not  had  the  opportunity  to  test  this 
question  by  means  of  the  cyanamin  and  neutral  red  reactions, 
but  hope  to  report  on  this  matter  in  the  near  future. 


BIBLIOGRAPHY. 
Bernard,  C. 

'59     Lecons  sur  les  proprietes  physiologiques  des  liquides  de  1'organisme,  Paris, 

P-  375- 
E  dinger. 

'79     Zur  Kenntnis  der  Driisenzellen  des  Magens,  besonders  beim  Menschen. 

Archiv  mikr.  Anat.,  17. 
Fitzgerald,  M.  P. 

'10     The  Origin  of  the  Hydrochloric  Acid  in  the  Gastric  Tubules.     Proc.  Roy. 

Soc.,  B.  83. 
Frankel. 

'91     Beitrage  zur  Physiologie  der  Magendriisen.     Archiv  f.  d.  gesammt.  Physiol. 
d.  Mensch.  u.  d.  Tiere,  48. 


FORMATION   OF   HYDROCHLORIC   ACID   IN   THE   FOVEOL^E.      249 

Greenwood,  M. 

'85     Observations  of  the  Gastric  Gland  of  the  Pig.     Journ.  of  Physiol.,  5,  p.  195. 
Heidenhain,  R. 

'70     Untersuchungen  iiber  den  Bau  der  Labdrtisen.     Archiv  f.  mikr.  Anat.,  6. 
'83     Die  Bildung  der  Satire  des  Magensaft.     Handbuch  Physiologic,  Hermann, 

5.  P-  149- 
Klemensiewicz,  R. 

'75     Uber  den  succus  pyloricus.     Sitzber.  d.  k.  Akad.  d.  wiss.,  math,  naturw. 

Klasse,  71,  Abth.  3,  p.  249. 
Langley. 

'81     On  the  Histology  and  Physiology  of  Pepsin-forming  Glands.     Phil.  Trans. 

Roy.  Soc.,  Lond.,  172,  part  3. 
Lepine,  R. 

'72     Recherches  experimentales  sur  la  question  de  savoir  si  certaines  cellules  des 
glandes    (dites   a    pepsine)    de    1'estomac   presentent    une   reaction   acide. 
Comptes  Rend.  Soc.  de  Biol.,  24. 
Macallum,  A.  B. 

'08     Die  Methoden  und  Ergebnisse  der  Mikrochemie  in  der  Biologischen  For- 

schung.     Ergebnisse  d.  Physiol.,  7  p.  628. 
Nencki  und  Pawlow. 

'96  Uber  dem  Ammoniakgehalt  des  Blutes  und  der  Organe  und  die  Harnstoff- 
bildung  bei  den  Saugetieren.  Archiv  f.  experiment.  Path,  und  Pharmakol. 
37.  P-  26. 

Nietzke,  R.,  and  Bossi,  A. 
'92     Zur  Kenntniss  der  Oxazinfarbstoffe.     2.  Einwirkung  von  Anilin  auf  Mel- 

dola's  Naphtolblau.     Berichte  d.  Deut.  chem.  Ges.,  Bd.  25. 
Rosemann. 

'07     Beitrage  zur  Physiologic  der  Verdauung.     Archiv  f.  d.  gesammt.  Physiol. 

d.  Mensch.  u.  d.  Tiere,  118. 
Sehrwald. 

'89     Die  Belegzellen  des  Magens  als  Bildungsstatten  der  Saure.      Miin.  Med. 

Wochens.,  n,  p.  177. 
Stohr,  P. 
'82     Zur   Kenntnis   des   feineren   Baues   der   menschlichen   Magenschleimhaut. 

Archiv  f.  mikr.  Anat.,  20. 
Witt,  O. 

'90  Uber  die  Cyanamine,  cine  neue  Gruppe  von  Farbstoffen.  Ber.  d.  Chem. 
Ges.,  23,  p.  2247. 


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